Investigating the origin of hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis> L.) cultivars using chloroplast microsatellites

The place and time of European hazel (Corylus avellana L.) domestication is not clear, although it was already cultivated by the Romans. In this study, 75 accessions from Spain, Italy, Turkey, and Iran were analysed using 13 chloroplast microsatellite to investigate the origin and diffusion of hazelnut cultivars. Four loci were polymorphic and identified a total of four different chlorotypes. Their distribution was not uniform in each geographical group. The most frequent chlorotype A was present in all groups. An increase in chlorotype number and diversity from Spain eastward to Italy, Turkey, and Iran was observed. Results suggest that some spread of cultivars occurred from East to West and that hazelnut cultivation was not introduced from the eastern Mediterranean basin into Spain and southern Italy by Greeks or Arabs. Moreover, the results suggest considerable exchange of germplasm between Italy and Spain, probably by the Romans. Hazelnut appears to have been domesticated independently in three areas: the Mediterranean, Turkey, and Iran.     

Size homoplasy and mutational behavior of chloroplast simple sequence repeats (cpSSRs) inferred from intra- and interspecific variations in four chloroplast regions of diploid and polyploid <Emphasis Type="Italic">Triticum</Emphasis> and <Emphasis Type="Italic">Aegilops</Emphasis> species

Chloroplast simple sequence repeats (cpSSRs) are widely distributed in the chloroplast genomes of all plant species, and are frequently employed for genotypic and phylogenetic analysis. However, information on intra- and interspecies variation in cpSSRs is lacking. In this study, we sequenced four intergenic (non-coding) chloroplast DNA regions in 57 accessions of 12 tetraploid, and 16 accessions of 4 hexaploid species of Triticum and Aegilops. These sequence data added to our previous data for diploid species in the same chloroplast regions. Intra- and interspecific genetic variation was analyzed for a total of 189 accessions of 13 diploid, 12 tetraploid, and 4 hexaploid species of Triticum and Aegilops, such that all species were represented by multiple accessions. The data were used to infer phylogenetic relationships within and among Triticum and Aegilops species. Based on this robust phylogenetic tree, seven of eight cpSSR loci clearly exhibited “size homoplasy,” referring to the fact that cpSSRs of identical size and DNA sequence can arise even if the alleles are not descended from a common ancestor. These data indicate that cpSSRs should be used with caution in phylogenetic analyzes. Interestingly, as observed from several previous studies, our data also suggest that observed mutation rates may increase significantly when mononucleotide (homopolymer) repeat numbers reach or exceed 9 bp. In the present report, using this sequence data set involving cpSSRs, 81 unique haplotypes among 189 accessions were detected, and five tetraploid Triticum and Aegilops species were successfully identified and genotyped. Our results indicate that combinations of nucleotide substitutions, indels and SSRs of chloroplast nucleotide sequences are available for genotyping at the species accession level.     

Chloroplast SSR genetic diversity indicates a refuge for Corylus avellana in northern Portugal

The genus Corylus, a member of the birch family Betulaceae, includes several species that are widely distributed throughout temperate regions of the Northern Hemisphere. The development of microsatellites or simple sequence repeats (SSRs) for non-coding regions of the chloroplast genome and their higher sequence variation compared with coding regions has provided a higher resolution tool for the study of cultivars and closely related taxa. Chloroplast polymorphisms provide a marker system to evaluate the genetic structure of plant populations. This study investigated genetic diversity in three cultivars and 32 genotypes of Corylus avellana L. from Portugal: 13 wild genotypes and 19 Portuguese landraces. Four of ten cpSSR loci were polymorphic, with diversity indices ranging from 0.111 to 0.244. Eleven chlorotypes were detected, and their relationships were analyzed using a network model. Haplotype A was most frequent in landraces and cultivars. Four chlorotypes (H, I, J and L) were found only in wild hazelnuts. The diversity of chlorotypes in the wild hazels, and the limited number reported in cultivars, suggests that northern Portugal was a refuge for hazel during the last ice age.     

Genetic diversity and origin of cultivated potatoes based on plastid microsatellite polymorphism

The origin of cultivated potatoes has remaining questions. In this study, 237 accessions of all cultivated species and 155 accessions of wild species closely related to cultivated potatoes, including their putative ancestors, were analyzed using 15 plastid microsatellites (SSRs) to investigate genetic diversity and their relationships with the wild species. We here used polymorphic plastid SSRs we developed from potato plastid genome sequences as well as already known plastid SSR markers. All 15 loci were polymorphic and identified a total of 127 haplotypes. Dramatic decreases in levels of genetic diversity were revealed in landraces in comparison with wild ancestor species. The plastid SSR results showed a decrease in haplotype number and diversity from Peru to both north and south. Phylogenetic analysis revealed two distinct groups. One of them, group A, contained the majority of accessions of cultivated species of the Solanum tuberosum Andigenum group including all accessions of cultivated diploid and triploid cytotypes of this group (S. chaucha, S. phureja, and S. stenotomum by a former taxonomic system) and most of tetraploid accessions of the S. tuberosum Andigenum group (S. tuberosum subsp. andigenum), and the majority of accessions of wild ancestors from the northern members of the S. brevicaule complex. Another group B comprised most of the wild species accessions and almost exclusively hybrid cultivated species which have introgressed plastid genomes from the other wild gene pools. Lack of clustering of traditional cultivated species (as used above) support a revised group classification of S. tuberosum.     

Analysis of genetic diversity in Indian robusta coffee genepool (<Emphasis Type="Italic">Coffea canephora</Emphasis>) in comparison with a representative core collection using SSRs and AFLPs

Genetic diversity among 40 accessions (Coffea canephora) of robusta coffee genepool available in India was determined in comparison with 14 representative samples from a C. canephora core collection and three accessions of C. congensis, using amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) markers. Both these molecular approaches were able to generate unique fingerprints for each of the accessions analysed. All the 12 SSR primers used in the present study were found polymorphic, with an average of six alleles per primer pair. Comparative analysis revealed the higher amount of diversity in representatives from a core collection than in the Indian genepool. Moreover, a total of 205 polymorphic AFLP bands were scored in all the 57 accessions analysed. The genetic relationship among 57 accessions was compared on the basis of SSR and AFLP polymorphisms. Genetic similarity dendrograms showed high correlation between the two marker systems. This study clearly established the high amount of diversity present in core samples, which is not represented in Indian genepool. Furthermore, the three accessions of C. congensis did not exhibit any significant diversity from other robusta accessions supporting the school of thought that C. congensis forms a biotype of C. canephora. The potential use of SSRs and AFLP markers in genetic diversity analysis for better ex situ management and also for exploitation of diversity in breeding programmes is discussed.     

Characterization of Seashore Paspalum (Paspalum vaginatum Swartz) Germplasm by Transferred SSRs from Wheat, Maize and Sorghum

One hundred and thirty SSR markers from wheat, maize and sorghum were screened for the transferability to Paspalum. The transfer rate was 67.5, 49.0 and 66.8% respectively. This would be a very efficient approach for DNA marker development for species which are not well studied molecularly. The polymorphism level for transferred SSR markers was 51.5% within species (Paspalum vaginatum) and 87.1% among Paspalum species. The high level of polymorphism is directly related to the high degree of heterozygosity maintained by its way of reproduction, i.e. self-incompatibility. Forty transferred polymorphic SSR markers were selected and used for characterization and evaluation of seventy-three Paspalum accessions. In total, 209 polymorphic bands were detected from these 40 SSR markers, with an average of five polymorphic bands per marker. The Paspalum accessions clustered into three major groups. Two very similar dendrograms can be generated from either 109 or 209 polymorphic bands. This led us to determine that 18 of the transferred SSR markers were sufficient for genetically differentiating the investigated germplasm accessions. The number of SSR markers required for germplasm characterization and evaluation is discussed. This is the first report of the transfer of SSR markers from major field crops to newly emerged environmental turfgrasses.     

The use of microsatellite polymorphisms to characterise and compare genetic variability in Avena strigosa and A. barbata

Microsatellite (SSR) polymorphism was assessed across 90 diploid Avena strigosa Schreb. and tetraploid Avena barbata Pott ex Link accessions obtained from the USDA-ARS National Small Grains Collection using 105 genomic SSRs. Eleven polymorphic SSRs that detected 69 different alleles were identified and used to genotype the 90 accessions, which were chosen from a larger set of 385 accessions based on geographical source-diversity and variable reaction responses to five Australian pathotypes of the crown rust pathogen Puccinia coronata Corda f. sp. avenae Eriks. Eight diploid and eight tetraploid clades were identified among the 90 accessions. Diploid accessions displayed the lowest genetic diversity, with all accessions being at least 86 % similar, and included accessions from countries in the Americas such as Canada, USA, Argentina, Uruguay and Brazil, and European accessions from France, Romania and Poland. Although both species formed distinct clusters in the dendrogram, a few instances of diploids showing high similarity with tetraploids and vice versa were observed. An AMOVA analysis revealed 86 % of the total genetic variation to be distributed within the two oat species, while between-species differences accounted for only 14 %. Heterozygosity (H) index values of 0.32 and 0.40 were obtained for diploids and tetraploids respectively. Our study effectively differentiated A. strigosa and A. barbata, and identified 11 SSRs suitable for future characterisation of accessions of the two species.     

Comparison of genetic variation among accessions of <Emphasis Type="Italic">Aegilops tauschii</Emphasis> using AFLP and SSR markers

Genetic diversity of 54 accessions of Aegilops tauschii from five countries was assessed using sequence-tagged microsatellites (or simple sequence repeats, SSRs) and amplified fragment length polymorphisms (AFLPs). In the case of AFLP analysis, a total of 256 amplification products obtained, 234 of them were polymorphic across all the 54 accessions. A total of 224 fragments were obtained from the 24 SSR primers and 219 of fragments were polymorphic across all the genotypes screened. Based on both AFLP and SSR markers, the highest percentage of polymorphisms were obtained in Iranian and accessions of unknown origin. The highest polymorphic information content (PIC) value was observed for SSRs (0.82) while the highest marker index (MI) value was for AFLPs (8.5) reflecting the hyper-variability of the first and the distinctive nature of the second system. Principal co-ordinate analysis (PCO) revealed congruent patterns of genetic relationships for both data sets, but did not group accessions strictly according to their geographical origins. Poor correlation was found between AFLP and SSR marker loci. This low association may be due to low number of AFLP and SSR markers. These results show that molecular markers can help to organize the genetic variability and expose useful diversity for breeding purposes.     

Genetic characterization of guava (Psidium guajava L.) germplasm in the United States using microsatellite markers

Genetic diversity of 35 Psidium guajava L. accessions and three related species (P. guineense Sw., P. sartorianum (O. Berg) Nied. and P. friedrichsthalianum (O. Berg) Nied.) maintained at the U.S. Department of Agriculture (USDA), National Plants Germplasm System, Hilo, HI, was characterized using 20 simple sequence repeat (SSR) markers. Diversity analysis detected a total of 178 alleles ranging from 4 to 16 alleles per locus. The observed mean heterozygosity (0.2) and inbreeding coefficient (0.8) indicated a high level of inbreeding among the accessions tested. Multi-locus DNA fingerprints based on the 20 SSR loci unambiguously differentiated all accessions and revealed the absence of duplicated samples. Ordination and cluster analyses suggested that the genetic relationships between majorities of the accessions could be explained by geographic origin, mainly including tropical America, Southeast Asia and Hawaii. A Bayesian cluster analysis partitioned the accessions into two groups and the partition was largely compatible with the result of ordination analyses. Distance-based cluster analyses further indicated that accessions from same geographical region or breeding programs grouped together in spite of the inter-regional exchange of germplasm. Accessions from Southeast Asia were dominantly white fleshed, whereas accessions from tropical America and Hawaii exhibited diverse flesh colors. The results also indicated that accessions from the same region were likely derived from a small number of common ancestors or progenitors. All 20 SSRs were transferable to P. guineense, P. sartorianum and P. friedrichsthalianum, indicating a close relationship between the cultigens and wild relatives. Application of SSR markers in the USDA/Agricultural Research Service germplasm collection provides new insight into the diversity of the guava germplasm, which will be valuable in future breeding endeavors and the conservation of guava genetic resources.     

Characterization of a world collection of <Emphasis Type="Italic">Agropyron cristatum</Emphasis> accessions

The genetic diversity was studied of 115 Agropyron cristatum accessions from 17 countries. Tetraploids were the most common (74.8%), followed by diploid (16.3%) and hexaploid (6.9%). We observed a relation between geographic distribution and ploidy level. The tetraploids, the most widespread, were found from Europe through Russia to East Asia. The diploids appeared over the same general range, except in Turkey, Iran and Georgia where no diploid accessions were found. Hexaploid accessions mainly came from a region comprising the east of Turkey, the north of Iran and Georgia. A selection of 71 accessions, including all three ploidy levels, were analyzed by capillary electrophoresis using six wheat simple sequence repeat (SSR) markers. All markers presented high levels of polymorphism, generating 166 different alleles ranging in size between 84 and 256 bp. Based on polymorphic information content values obtained (0.579–0.968), all the SSRs were classified as informative markers (values?>?0.5). According to the dendrogram generated, all the A. cristatum accessions were distinctly classified. Diploid, tetraploid and hexaploid accessions are not clearly differentiated from each other on the basis of SSR markers. A field experiment was conducted to morphologically characterize 18 accessions including the three ploidy levels. Significant differences were found between the accessions in spike length, spike width and number of spikelets per spike. All the cytological, molecular, and morphological data demonstrate the high genetic diversity present in A. cristatum, making it a valuable resource for future breeding programs.     

Using diversity of the chloroplast genome to examine evolutionary history of wheat species

Chloroplast microsatellites (SSRs) are conserved within wheat species, yet are sufficiently polymorphic between and within species to be useful for evolutionary studies. This study describes the relationships among a very large set of accessions of Triticum urartu Thum. ex Gandil., T. dicoccoides (Körn. ex Asch. et Graebn.) Schweinf., T. dicoccon Schrank, T. durum Desf., T. spelta L., and T. aestivum L. s. str. based on their cpSSR genotypes. By characterising the chloroplast diversity in each wheat species in the evolutionary series, the impact on diversity of major evolutionary events such as domestication and polyploidyisation was assessed. We detected bottlenecks associated with domestication, polyploidisation and selection, yet these constrictions were partially offset by mutations in the chloroplast SSR loci that generated new alleles. The discrete cpSSR alleles and haplotypes observed in T. urartu and Aegilops tauschii, combined with other species specific polymorphisms, provide very strong evidence that concur with current opinion that neither species was the maternal and thus cytoplasmic donor for polyploid wheats. Synthetic hexaploid wheats possessed the same chloroplast haplotypes as their tetraploid progenitors demonstrating how the novel synthetic wheat lines have captured chloroplast diversity from the maternal parents, the chloroplast is maternally inherited and novel alleles are not created by genomic rearrangements triggered by the polyploidisation event.     

Characterization of American hazelnut (Corylus americana) accessions and Corylus americana?×?Corylus avellana hybrids using microsatellite markers

The American hazelnut (Corylus americana Marshall) is native to the eastern United States and Southern Canada. In the early decades of the last century, breeding work by several individuals attempted to combine the cold hardiness and disease resistance of the American hazelnut with the larger nut size of the European hazelnut (C. avellana L.). Many hybrid selections grown today trace back to these early efforts. Over the past three decades, representatives of C. americana were collected and are preserved in Corvallis, Oregon. Seeds were collected along roadsides, in hedgerows, in woodland clearings, and in gardens. The seeds were germinated and the best of the resulting seedlings were preserved for later use. In this study, genetic diversity was studied in 87 American and 67 hybrid hazelnut selections using 21 microsatellite loci. The total number of alleles was 266 in the 162 accessions examined, of which 229 were present in C. americana accessions, but only 168 in the Arbor Day Farm hybrids, and 116 in the hybrids of C. americana ‘Rush’. In the American accessions, a high level of genetic diversity was observed (H _e ?=?0.74, H _o ?=?0.68). A genetic similarity matrix of the American and hybrid accessions, and a few European cultivars, was constructed and the resulting dendrogram revealed seven major groups: European cultivars and ‘Rush’ hybrids, two groups of Arbor Day Farm hybrids, three groups of C. americana accessions, and a mixed group of hybrid and American accessions. Analysis confirmed the reported parentage of 10 hybrids. The genetic diversity among the American hazelnut accessions, untapped to date, will be useful in breeding hybrids that will allow expansion of hazelnut production into eastern North America.     

Characterization of European hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis>) cultivars using SSR markers

World hazelnut production is based primarily on selections from the wild. In this study, we used 21 pairs of simple sequence repeat (SSR) primers to investigate genetic diversity in 270 clonal accessions of European hazelnut (Corylus avellana) representing a wide geographic range. Of the 270 accessions, 198 had unique fingerprints while 72 were duplicates. Based on the 198 unique accessions, the number of detected alleles per locus averaged 9.81 and observed heterozygosity (H_o) averaged 0.67. Of the 206 total alleles amplified, 20 were unique to a single accession. A genetic similarity matrix was constructed and the resulting dendrogram revealed four major geographical groups: Central European, Black Sea, English and Spanish-Italian. SSR alleles indicated the parentage of 31 accessions. The fingerprints are publicly available through the Germplasm Resources Information Network (GRIN) database. The identification of duplicate and mislabeled accessions will improve management of hazelnut genebanks, and information on genetic variation in hazelnut will assist the international research community. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

EST <Emphasis Type="Italic">versus</Emphasis> Genomic Derived Microsatellite Markers for Genotyping Wild and Cultivated Barley

Genetic variation present in wild and cultivated barley populations was investigated using two sources of microsatellite also known as simple sequence repeat (SSR) markers. EST-SSRs are derived from expressed sequences and genomic SSRs are isolated from genomic DNA. Genomic SSR markers detected a higher level of polymorphism than those derived from ESTs. Polymorphism information content was higher in genomic SSRs than EST-derived SSRs. This study showed that the EST-SSR markers developed in cultivated barley are polymorphic in wild and cultivated varieties and produced high quality markers. Ten of these functional markers were polymorphic across the accessions studied. EST markers indicated clearer separation between wild and cultivated barley than genomic SSRs. The EST-SSRs are a valuable source of new polymorphic markers and should be highly applicable to barley genetic resources, providing a direct estimate of functional biodiversity.     

Discriminating power of microsatellites in cranberry organelles for taxonomic studies in <Emphasis Type="Italic">Vaccinium</Emphasis> and Ericaceae

Simple sequence repeats (SSRs) in chloroplast and mitochondrial DNA, which have not been previously developed in the Ericaceae or, more specifically, in the genus Vaccinium, can be powerful tools for determining evolutionary relationships among taxa. In this study, 30 chloroplast, 23 mitochondrial, and 1 mitochondrion-like SSRs were identified in cranberry (V. macrocarpon), and primer-pairs were developed and tested for each locus. Although no polymorphisms were detected for any of the 54 SSR loci in nine diverse cranberry genotypes, all primers were cross-transferable to some extent to a panel of 12 additional Vaccinium taxa and four non-Vaccinium Ericaceae species. A Neighbor-Joining tree of the estimated average squared distances resolved the species by genus and by section within Vaccinium. Similar topologies with increased branch support were observed in Bayesian inference trees constructed from the DNA sequences of six plastid and two mitochondrial SSR loci. Two multiplexing/poolplexing panels of M13 fluorescently labeled primers, which amplify 24 of the 54 markers, were developed and can serve as an efficient, cost-effective means for characterizing the basic molecular phylogeny of Vaccinium. Increased understanding of evolutionary relationships among Vaccinium species should facilitate interspecific hybridization and introgression efforts to improve economically important traits of commercial berry crops.     

Exploitation of <Emphasis Type="Italic">Malus</Emphasis> EST-SSRs and the utility in evaluation of genetic diversity in <Emphasis Type="Italic">Malus</Emphasis> and <Emphasis Type="Italic">Pyrus</Emphasis>

A total of 8117 suitable SSR-contaning ESTs were acquired by screening from a Malus EST database, among which dinudeotide SSRs were the most abundant repeat motif, within which, CT/TC followed by AG/GA were predominant. Based on the suitable sequences, we developed 147 SSR primer pairs, of which 94 pairs gave amplifications within the expected size range while 65 pairs were found to be polymorphic after a preliminary test. Eighteen primer pairs selected randomly were further used to assess genetic relationship among 20 Malus species or cultivars. As a result, these primers displayed high level of polymorphism with a mean of 6.94 alleles per locus and UPGMA cluster analysis grouped twenty Malus accessions into five groups at the similarity level of 0.6800 that were largely congruent to the traditional taxonomy. Subsequently, all of the 94 primer pairs were tested on four accessions of Pyrus to evaluate the transferability of the markers, and 40 of 72 functional SSRs produced polymorphic amplicons from which 8 SSR loci selected randomly were employed to analyze genetic diversity and relationship among a collection of Pyrus. The 8 primer pairs produced expected bands with the similar size in apples with an average of 7.375 alleles per locus. The observed heterozygosity of different loci ranged from 0.29 (MES₉₆) to 0.83 (MES₁₃₈), with a mean of 0.55 which is lower than 0.63 reported in genome-derived SSR marker analysis in Pyrus. The UPGMA dendrogram was similar to the previous results obtained by using RAPD and AFLP markers. Our results showed that these EST-SSR markers displayed reliable amplification and considerable polymorphism in both Malus and Pyrus, and will contribute to the knowledge of genetic study of Malus and genetically closed genera.     

EST-SSR cross-amplification and genetic similarity in <Emphasis Type="Italic">Onobrychis</Emphasis> genus

EST-SSR from Medicago truncatula Gaertn. and Glycine max (L.) Merr. were tested for transferability in various species of Onobrychis (O. pyrenaica Sennen, O. argentea Boiss. and O. viciifolia Scop.). Repeatable amplification was obtained for 81% of the microsatellites and 52% were polymorphic. Six selected SSRs from M. truncatula were used to fingerprint and estimate the genetic similarity of a set of 23 accessions of O. viciifolia. PCA analysis discriminated among the different Onobrychis species and the sainfoin accessions were clustered in a single major group. This grouping is discussed in terms of the history of cultivation of sainfoin in Spain. The selected SSRs will allow fingerprinting and genetic studies in Onobrychis species, solving the lack of available SSR markers in this species.     

Development of EST-SSR in foxtail millet (<Emphasis Type="Italic">Setaria italica</Emphasis>)

The development of EST-SSR in foxtail millet (Setaria italica) for polymorphism and transferability study was reported here. From 1213 EST sequences, 30 SSRs were obtained and primers were designed for 26 SSRs. Among them, four pairs of SSR primers amplified polymorphic products in 12 foxtail millet cultivars and one accession of Setaria viridis, a wild relative of foxtail millet, with 10 alleles detected for the four loci and 2.5 alleles per locus. In addition, ten SSR markers could be transferred to other nine Gramineae species. The putative functions of 11 ESTs containing polymorphic and transferable SSRs were also identified.